1. Field of the Invention
The present invention relates to a method and apparatus for assay in utilizing attenuated total reflection. More particularly, the present invention relates to a method and apparatus for assay in utilizing attenuated total reflection in which a sensing surface in a flow channel can be washed sufficiently and rapidly.
2. Description Related to the Prior Art
An assay apparatus for assay in utilizing attenuated total reflection is used for various kinds of studies in a biochemical field or the like, for example to study interaction of protein, DNA and various biomaterials, and to select candidate drugs by screening. Also, the technique is useful in the fields of the clinical medicine, food industries and the like.
A surface plasmon resonance (SPR) sensor is known as an assay apparatus in utilizing attenuated total reflection. A thin film/dielectric interface of a metal film is fitted on a dielectric block. Light is directed to the thin film/dielectric interface in a manner conditioned for total reflection. Surface plasmon is a term to mean the compressional wave created on the surface of the metal and included in plasmon as quantized expression of the compressional wave. Free electrons in a metal vibrate to generate the compressional wave.
The assay apparatus includes a light source device and a photo detector. The light source device emits light beams to the interface in the sensor unit at plural incident angles to satisfy the total reflection condition. The photo detector receives the reflected light from the interface, and detects intensity of the reflected light. The sensor unit has the thin film of metal. In the assay apparatus, surface plasmon resonance (SPR) is generated on the sensing surface. Reaction or interaction of a sample is assayed by measuring the surface plasmon resonance. An example of this is disclosed in JP-A 2002-310903.
Illuminating light is applied to an interface between the thin film and the prism or a surface back to the sensing surface at an angle of incidence equal to or more than a critical angle to satisfy a condition of total reflection. Then total reflection of the illuminating light occurs. Upon the total reflection created on the metal/dielectric interface, a small component of the light passes through the metal film without reflection, and penetrates to the sensing surface. A wave of the penetrating component is called an evanescent wave. Surface plasmon resonance (SPR) is created when frequency of the evanescent wave coincides with that of the surface plasmon. In response to this, intensity of the reflected light attenuates remarkably. In the assay apparatus, the attenuation in the reflected light reflected by the metal/dielectric interface is detected, to recognize creation of the SPR on the sensing surface. Intensity of the reflected light incident at an incident angle or resonance angle for creating the SPR is attenuated, so as to form a dark line on a photo reception surface.
A resonance angle or an angle of incidence of light for creation of surface plasmon resonance depends upon a refractive index of a medium of transmission of evanescent waves and surface plasmon. In other words, a change in the refractive index of the medium of transmission causes a change in the resonance angle of creation of SPR. The substance or sample in contact with the sensing surface is the medium for transmitting the evanescent waves and surface plasmon. When binding, dissociation or other reaction occurs on the sensing surface between two molecules or samples, the resonance angle changes because of a change in the refractive index of the medium of transmission. The SPR assay apparatus finds the changes in the resonance angle, to assay the interaction between the molecules or samples.
An assay apparatus for assay in utilizing attenuated total reflection is used for various kinds of studies in a biochemical field or the like, for example to study interaction of protein, DNA and various biomaterials, and to select candidate drugs by screening. In the screening, a ligand is protein or biomaterial. An analyte is any one of plural drugs, and is caused to contact the ligand for the purpose of investigating interaction between those. Also, the technique is useful in the fields of the clinical medicine, food industries and the like.
In an assay step for measuring interaction, at first analyte fluid containing analyte is caused to flow on a sensing surface where ligand is immobilized. So the ligand is contacted by the analyte in the measurement. After this, liquid buffer is introduced to the sensing surface, to measure reaction of dissociation in which the analyte is dissociated from the ligand. An assay signal in the reaction is detected in a real time manner in the assay step, and is recorded as measuring data.
It is important to increase throughput or efficiency of the assay particularly for the purpose of assaying reaction of a great number of samples. A reaction speed varies between the samples. A first one of the samples with a high value of the reaction speed can be assayed for obtaining a result of reaction such as presence or existence of binding. A second one of the samples with a low value of the reaction speed will take considerable time for obtaining a result of reaction. If a measuring time between the start and end of the assay is kept unchanged between samples, the measuring time is excessively long for a first one of the samples of which the reaction speed is high. The measuring time is excessively short for a second one of the samples of which the reaction speed is low.
JP-A2002-310903 discloses an assay method in which an assay signal output by the assay is monitored, so as to determine the measuring time according to the signal. The measuring time can be adjusted and minimized according to the reaction speed and the samples, so as to increase the throughput or efficiency of the assay.
After the assay step, the sensing surface is washed and regenerated by a washing step for a predetermined washing time. Washing liquid or regenerant is introduced to the sensing surface to remove residual part of the analyte remaining on the sensing surface. The sensing surface being regenerated is used in the assay.
However, there is a shortcoming of the known method due to a constant value of the predetermined washing time for the sensing surface. Should the washing time be too short, the washing is incomplete. Part of the analyte remains on the sensing surface, and influences measuring data of next assay, to lower precision in the assay. Should the washing time be too long, the throughput or efficiency cannot be high.